Photodynamic therapy (PDT) involves the systemic administration of a tumor localizing photosensitizing drug that is relatively benign in the absence of light. However, photoactivation of the sensitizer in the presence of oxygen generates highly reactive and cytotoxic molecular species through various photophysical pathways (i.e. Type I or Type II mechanisms) which have the ability to destroy malignant tissue (Kessel, D. Photochem. Photobiol. 39:851-859, 1984; Foote, C. S. Science 162:963-970; Gomer, C. J. Photochem. Photobiol. 54:1093-1107; Moan, J. et al., Photochem. Photobiol. 55:931-948, 1992). Since tissue injury requires the simultaneous presence of both photosensitizer and light, spatial confinement of the photoirradiation to the proximity of the tumor imparts a dual selectivity to the treatment. This enhanced selectivity results in a modality of cancer treatment with minimal side effects and thus represents a major advantage over the more common therapeutic approaches (surgery, chemotherapy and ionizing radiation therapy) in use today. Currently, world wide clinical investigations of PDT are being carried out with Photofrin.TM. and HPD. Photofrin.TM. has recently been approved for use by health agencies in Canada (prophylaxis of recurrent papillary bladder tumors), the Netherlands (early and late stage lung cancer and for obstructing esophageal tumors) and Japan (early stage lung, esophagus, bladder, gastric and cervical cancers). Although encouraging results have been obtained with these photosensitizing agents for a wide variety of tumors, it appears that the following limitations may adversely affect the therapeutic outcome of PDT: (a) a low absorption coefficient in the region where activating light penetrates tissue most efficiently (600-900 nm) limits the depth of PDT damage in large tumors; (b) acute effects leading to rapid vascular occlusion and subsequent loss of oxygen supply are primarily responsible for tumor eradication, not direct tumor cell killing; because photodynamic processes are dependent on oxygen, this rapid shift of cells into hypoxia may limit the further photodestruction of a fraction of the tumor cells (Henderson, B. W. et al., Photochem. Photbiol. 49:299-304, 1989; Tromberg, B. J. et al., Photochem. Photobiol. 52:375-385, 1990) and (c) prolonged retention of these drugs in the skin leads to dermal photosensitivity for months following treatment.
The successes of PDT has fostered the search for and development of "second generation" photocytotoxic compounds in the hopes of increasing the efficacy of the treatment and minimizing the limitations displayed by the drugs currently in use. The in vitro and in vivo use of PDT employing 5-ethylamino-9-diethylaminobenzoa!phenothiazinium chloride (EtNBS) have been under investigation (Cincotta, L. et al., Photochem. Photobiol. 46:751-758, 1987; Cincotta, A. H. et al., SPIE Proceedings 1203:202-210, 1990; Cincotta, L., et al., Cancer Res. 53:2571-2580, 1993; Cincotta, L., Cancer Res. 54:1249-1258, 1994). This novel photosensitizer possesses several characteristics which differentiate it from the porphyrin family of drugs (including Photofrin.TM. and HPD), the largest class of photosensitizers investigated for PDT. First, EtNBS is easily synthesized as a single, pure compound. Second, the rapid intracellular accumulation of EtNBS leads to tumor destruction primarily through direct cell killing with minimal effects to the vasculature. Third, it absorbs light (652 nm) very efficiently (extinction coefficient 20.times.Photofrin.TM.) in the "therapeutic window". Fourth, the drug is essentially eliminated from the majority of murine tissues 24 hours after its administration and importantly, insignificant damage occurs to the surrounding normal skin following PDT of subcutaneous tumors.
Benzophorphyrin derivative mono-acid A ring (BPD-MA), disclosed in Levy et al., U.S. Pat. No. 4,920,143 has also shown promise in studies on photodynamic therapy of tumors, it being reported that BPD-MA is a more effective phototoxic agent than hematoporphyrin (HPD) (Richter, A. M. et al., JNCI 79:1327-1332, 1987).
In order to further increase the selectivity and/or efficacy of cancer treatments, approaches using various combinations of surgery, radiotherapy, chemotherapy, hyperthermia, and more recently immunotherapy are becoming more prevalent. These other therapies have been investigated in conjunction with PDT in order to enhance tumor eradication and PDT has been successfully combined with many of these other therapies. There have, however, been only a limited number of studies in which researchers used a combination of photosensitizers in order to achieve enhanced PDT effects. The choice of drugs in these studies was usually based on an attempt to exploit differences in the photosensitizers' mechanism(s) of action or cellular site(s) of toxicity. Nelson et al., JNCI 82:868-873, (1990) studied the combination of Photofrin II and meso-tetra-(4-sulfonatophenyl)-porphine) (TPPS4), which typically acts by direct cell killing, with the EMT-6 tumor model. In this study the treated tumors were 5-7 mm in diameter; larger tumors gave unsatisfactory results, i.e. they did not respond significantly to treatment.
Nelson et al. found that the effects observed were not the result of two different mechanisms of action (vasculature shut down and direct intracellular effects) but solely the result of damage to the microvasculature by both compounds.
Johnson et al., Abst. 21st Ann. Mtng. Am. Soc. Photobiol. MAM-F6, 47S-48S, (1994), examined the effects resulting from the combination of an anionic and cationic photosensitizer, hexyl pyropheophorbide hexyl ether and Victoria Blue-BO, respectively, on Colon 26 tumors. Their data implies that PDT with the dual photosensitizers gave an enhanced response and also that the combined response was the result of each drug eliciting a different photoeffect. When applied to relatively small tumors (30-40 mm.sup.3), this combination phototherapy resulted in a cure rate of 23%.
Foultier et al., J. Photochem. Photobiol. B. Biol. 10:119-132, (1991), investigated the multidrug treatment of L1210 leukaemic cells using two photosensitizers, hematoporphyrin derivative and rhodamine 123, that localize to different parts of cells. The combination therapy did not increase cell toxicity except at high drug doses.
The method of the instant invention differs from the above mentioned combination PDT protocols in several important ways. For example, the tumors which can be successfully treated in mouse tumor models by the present invention are large (8-10 mm) compared to the tumors (5-7 mm) usually treated in the PDT literature, and yet 76% were eradicated. Large tumors have a history of poor response to PDT; as a result, the majority of the prior investigations using either EtNBS or BPD-MA have been carried out on tumors less than 7 mm in diameter. Previous studies by the present inventors have shown that large tumors initially respond to EtNBS-PDT (eschar formation, suppression of growth) but they match the control tumor weights at 2 weeks post-PDT. The relationship between tumor volume and response to HPD-PDT using the RIF (radiation induced fibrosarcoma) animal model has also shown a direct correlation between tumor size and rate of regrowth (Al-Watban, A. H. F., Surgery and Medicine 10:165-172, 1990). It is accepted by the art that the incomplete tumor destruction observed when treating large tumors results from an inadequate penetration of the tumor by the activating light source.
Another difference between the method of the present invention and prior art methods are the unexpected and surprising results achievable by the invention. Histological examination of the EtNBS/BPD-MA treated tumors 24 h post PDT showed virtually no damage to the vasculature, as evidenced by little if any extravasation of red blood cells, no stasis, no damage to the surrounding normal tissue (skin), but nearly total damage to the tumor mass (cells were highly pyknotic). There was also a substantial decrease in the total tumor cell density following PDT. The fact that the 24 h histology showed no visible damage to the tumor vasculature was surprising, since it had been hoped that it would be possible to exploit the vascular-destructive mode of action of BPD-MA to enhance the PDT effect.
Thus the present invention pertains to the combined use of EtNBS with a promising new photosensitizer known as benzoporphyrin derivative monoacid ring A (BPD-MA) in an attempt to enhance PDT efficacy. As stated above, EtNBS appears to eradicate tumors primarily by direct tumor cell killing with minimal effects to the vasculature. Conversely, the work of Richter et al., Br. J. Cancer 63:87-93, (1991), showed that BPD-MA appears to primarily destroy solid tumors indirectly by the rapid occlusion of the microvasculature and the ensuing hypoxia. BPD-MA was used because it is activated by a wavelength of light (690 nm) that penetrates tissue to a greater depth than Photofrin.TM. (630 nm) and has been reported to be considerably more phototoxic to cancer cells in vitro (Richter, A. M., et al., JNCI 79:1327-1332, 1987). Also, the pharmacokinetics of drug uptake by the tumor is similar to EtNBS, i.e. parenteral administration results in the rapid accumulation of BPD-MA in the tumor. In addition, it is retained in the skin for a significantly shorter period of time compared to Photofrin.TM.. The surprisingly enhanced PDT effect of the combination of EtNBS and BPD-MA over either chromophore alone is illustrated by the treatment of large tumors (8-10 mm), which have been shown in preliminary investigations not to respond well to phototherapy with either chromophore alone (Cincotta, L., et al., Cancer Res. 54:1249-1258, 1994; Cincotta, L., et al. Photochem. Photobiol. 46:751-758, 1987).
The method of the present invention, treatment of mammals with the photosensitizer combination of EtNBS and BPD-MA and light, is the first to demonstrate a consistent eradication of large (8-10 mm) murine tumors with PDT. As indicated above, it is generally acknowledged in the art that PDT with a variety of photosensitizers is ineffective in eradicating tumors in mouse model systems once the diameter increases above 5-7 mm. Not only is the effect of the instantly claimed combination treatment much more dramatic than the result obtained from doubling the light dose or the concentration of either photosensitizer alone (implying a synergistic rather than additive effect); it was found that a higher BPD-MA dose (5.0 mg/kg) resulted in the significant death (77%) of animals treated with PDT. Therefore, in the animal model, the combined therapy of the present invention also increases the therapeutic index of phototreatment dramatically (i.e. there were zero deaths). The phenomenon of acute toxicity in animals with other modes of PDT has been reported previously (Dougherty, T. J., Photochem. Photobiol. 45:879-889, 1987; Cincotta, L., et al., Cancer Res. 54:1249-1258, 1994; Ferrario, A., et al., Cancer Res. 50:539-543, 1990).
Thus, it has now been surprisingly and unexpectedly discovered that the growth arrest or eradication of tumors that can be achieved by using only EtNBS as the PDT agent can be significantly augmented by simultaneously administering the PDT agent BPD-MA. It has been discovered that the effect of combining these two photosensitizers is synergistic rather than additive, allowing lower doses of each chromophore to be used and improving the therapeutic index of the chromophores. No damage to photoirradiated skin is found when dosages effective for tumor killing are used.
This discovery was entirely unexpected because the mechanism of action of tumor eradication using the combination of dyes is inconsistent with the known mechanisms of action of BPD-MA by itself, which is occlusion of tumor vasculature and subsequent tumor hypoxia. Nothing in the prior art suggested that the use of BPD-MA with EtNBS would increase the tumor cell killing effect associated with EtNBS while negating the vasculature-occluding effects previously associated with BPD-MA.